Ion reactions typically involve the reaction of either a positively or negatively charged ion with another charged species, which can be another positively or negatively charged ion or an electron. In electron induced dissociation (EID), for example, an electron is captured by an ion which can result in the fragmentation of the ion. EID has been used to dissociate bio-molecules in mass spectrometry (MS), and has provided capabilities that cover a wide range of possible applications from regular proteomics in liquid chromatography-mass spectrometer/mass spectrometer (LC-MS/MS) to top down analysis (no digestion), de novo sequencing (abnormal amino acid sequencing finding), post translational modification study (glycosylation, phosphorylation, etc.), protein-protein interaction (functional study of proteins), and also including small molecule identification.
After the first report of electron capture dissociation (ECD) using electrons having kinetic energies of 0 to 3 eV, other electron induced techniques have also been developed including electron transfer dissociation (ETD) (using reagent anions), Hot ECD (electrons with kinetic energy of 5 to 10 eV), electron ionization dissociation (EID) (electrons with kinetic energy greater than 13 eV), activated ions ECD (AI-ECD), electron detachment dissociation (EDD) (electrons with kinetic energy greater than 3 eV on negative ions), negative ETD (using reagent cations), and negative ion ECD (niECD, using electrons on negative ions), Electron Impact Excitation of Ions from Organics (EIEIO, electrons with kinetic energy greater than 3 eV on singly charged cations). ECD, ETD, Hot ECD, AI-ECD, and EIEIO are utilized for positively charged precursor ions, while others such as niECD are utilized for negatively charged precursor ions. EID can be utilized to dissociate precursor ions of both polarities, including singly charged precursors. Since their discovery, these ion reaction techniques have become very useful for analyzing biomolecular species, such as peptides, proteins, glycans, and post translationally-modified peptides/proteins. ECD, for example, allows top down analysis of proteins/peptides and de novo sequencing of them. Proton transfer reactions (PTR) can also be utilized to reduce the charge state of ions in which a proton is transferred from one charged species to another.
These electron induced dissociations are considered to be complimentary to conventional collision induced or activated dissociations (CID or CAD) and have been incorporated in advanced MS devices.
In ECD, low energy electrons (typically <3 eV) are captured by positive ions. Historically, ECD was performed in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR) because FT-ICR generally utilizes a static electro-magnetic field for ion confinement that avoids the heating of free electrons. However, such devices required relatively long interaction times and involved large instruments that were expensive to build. Attempts to use ECD in smaller applications involving Radio Frequency (RF) ion traps have been found to cause acceleration of electrons due to the trapping RF field. As a result, the field generally turned to ETD, which used negatively charged reagent ions as the electron source, or implemented ECD in a linear RF ion trap with a magnetic field.
The usage of the term ECD in the present teachings hereinafter should be understood to encompass all forms of electron related dissociation techniques, and is not limited to the usage of ECD with electrons with kinetic energy of 0 to 3 eV. Rather, usage of the term ECD within the present teachings is representative of electron related dissociation techniques, and should be understood to include all forms of electron related dissociation phenomenon including hot ECD, EID, EDD, EIEIO, and negative ECD.
ECD and ETD conventionally require relatively long reaction times between the precursor ions and reagent ions to effect (“de-ionization” or ionization)/dissociation. Though devices have been described that perform ion reactions in a “flow through” mode in which precursor ions are continuously flowed through the reaction region, such devices typically suffer from poor reaction efficiency. For example, it was reported that ExD product ion signal/total precursor ion signal can be less than 1%. See Voinov, V. G.; Deinzer, M. L.; Barofsky, D. F. Anal. Chem. 2009, 81, 1238-1243. Accordingly, linear ion traps have been utilized to simultaneously trap precursor and reagent ions during the ion reaction events, for example, with the electron injection and precursor ion injection/extraction sharing the same ports (or the same end lens electrodes). Trapping operations, however, typically require multiple steps and have poor compatibility with conventional CID-based quadrupole Time-of-Flight mass spectrometers (qToF), which generally operate in a continuous flow through manner. Moreover, the duty cycle is decreased because when trapping one analyte ion population, the rest of the analytical ion beam goes unused.
Recently, a new ECD device was reported that utilizes a branched RF ion trap structure in which a low-energy electron beam can be injected orthogonally into the analytical ion beam with independent control of both the ion and electron beams. See PCT App. No. PCT/IB2014/00893, filed on May 29, 2014, which is incorporated herein by reference in its entirety. This device could operate in either “flow-through” mode or simultaneous trapping mode, though it was reported that a short ion trapping period at the region of precursor ion and electron beam intersection could increase ECD efficiency while still providing up to five ECD spectra per second when operating in an information-dependent acquisition workflow.
Accordingly, there remains a need for ECD devices and methods for operating in a pure “flow through” mode, with high ion reaction efficiency.